Synthesis of Anti-Inflammatory Spirostene-Pyrazole Conjugates by a Consecutive Multicomponent Reaction of Diosgenin with Oxalyl Chloride, Arylalkynes and Hydrazines or Hydrazones

Steroid sapogenin diosgenin is of significant interest due to its biological activity and synthetic application. A consecutive one-pot reaction of diosgenin, oxalyl chloride, arylacetylenes, and phenylhydrazine give rise to steroidal 1,3,5-trisubstituted pyrazoles (isolated yield 46–60%) when the Stephens–Castro reaction and heterocyclization steps were carried out by heating in benzene. When the cyclization step of alkyndione with phenylhydrazine was performed in 2-methoxyethanol at room temperature, steroidal α,β-alkynyl (E)- and (Z)-hydrazones were isolated along with 1,3,5-trisubstituted pyrazole and the isomeric 2,3,5-trisubstituted pyrazole. The consecutive reaction of diosgenin, oxalyl chloride, phenylacetylene and benzoic acid hydrazides efficiently forms steroidal 1-benzoyl-5-hydroxy-3-phenylpyrazolines. The structure of new compounds was unambiguously corroborated by comprehensive NMR spectroscopy, mass-spectrometry, and X-ray structure analyses. Performing the heterocyclization step of ynedione with hydrazine monohydrate in 2-methoxyethanol allowed the synthesis of 5-phenyl substituted steroidal pyrazole, which was found to exhibit high anti-inflammatory activity, comparable to that of diclofenac sodium, a commercial pain reliever. It was shown by molecular docking that the new derivatives are incorporated into the binding site of the protein Keap1 Kelch-domain by their alkynylhydrazone or pyrazole substituent with the formation of more non-covalent bonds and have higher affinity than the initial spirostene core.

Taking into account the interest in diosgenin and pyrasole derivatives as anti-inflammatory agents, the effect of selected compounds was investigated using a histamine-induced mice paw edema model. The molecular docking of obtained pyrazole-diosgenin conjugates into the binding site of the protein Keap1 Kelch-domain was carried out.
Taking into account the interest in diosgenin and pyrasole derivatives as anti-inflammatory agents, the effect of selected compounds was investigated using a histamineinduced mice paw edema model. The molecular docking of obtained pyrazole-diosgenin conjugates into the binding site of the protein Keap1 Kelch-domain was carried out.
Initially, we worked out a three-component synthesis of alkyne-1,2-diones of diosgenin 1. Diosgenin 1 was first converted to the steroidal monooxalyl chloride 2 by treating with an excess of oxalyl chloride in chloroform at 0 • C. After removing the excess of oxalyl chloride and chloroform in vacuo, the resulting 3β-O-(2-chloro-2-oxo-aceta-te)-5-spirostene 2 (Scheme 2) was involved in the Stephens-Castro cross-coupling reaction with aryl alkynes (3a-e). It was known that cross-coupling of terminal acetylenes with monooxalyl chloride giving acetylenic diketones is best performed in ethereal solvent in the presence of CuI (5% mol) and Et 3 N (2 mmol) in ligand-free conditions. The Stephens-Castro reaction of steroidal oxalyl chloride 2 and phenylacetylene 3a in THF in the mentioned conditions led to compound 4 in a yield of 9%. Varying amounts of copper iodide (5, 10, 20 mol %) and Et 3 N (2 or 3 equiv.) and also using the additive (TMEDA, 0.1 equiv.) gave no acceptable results; the alkyne-1,2-dione 4 was obtained in trace amounts. Further, it was not possible to obtain the target compound 4 when replacing the base with N,N-diisopropylethylamine (DIPEA) or K 2 CO 3 , and the solvent with 1,4-dioxane. The best result for the coupling step was obtained by replacing ether solvents with benzene, reducing the amount of Et 3 N to 1 equiv. and increasing the amount of catalyst to 10 mol %. By performing the reaction at 40 • C, compound 4 was isolated in 48% yield after column chromatography. Increasing the reaction temperature diminishes the yield, and prolonging the reaction time (more than 24 h) does not increase the yield. Under the found conditions, spirostene ynediones 5-8, containing fragments of aryl acetylenes with 4-methoxy-, 4-fluoro-, 4-ethyland 3-methyl substituent, were obtained in 18-48% isolated yields. Characteristically, the content of alkyne-1,2-diones 4-8 in the reaction mixture was 70-80% according to 1 H NMR data; however, a significant part of the products was decomposed during isolation by chromatography on silica gel due to the low stability of alkyne-1,2-diones. The structure of (22R,25R)-spirost-5-en-3β-yl 2-oxo-4-(phenyl)but-3-ynoate 4 has been corroborated by an X-ray structure analysis ( Figure 1). synthesis of these heterocycles included the sequence of glyoxylation-alkynylation o yl(hetaryl) substrates [38,44] or activation-alkynylation of (hetero)arylglyoxylic acids the heterocyclization reaction of the resulting alkynyl diketones [39,40].
Initially, we worked out a three-component synthesis of alkyne-1,2-diones of d genin 1. Diosgenin 1 was first converted to the steroidal monooxalyl chloride 2 by tr ing with an excess of oxalyl chloride in chloroform at 0 °C. After removing the exces oxalyl chloride and chloroform in vacuo, the resulting 3β-O-(2-chloro-2-oxo-ac te)-5-spirostene 2 (Scheme 2) was involved in the Stephens-Castro cross-coupling r tion with aryl alkynes (3a-e). It was known that cross-coupling of terminal acetyle with monooxalyl chloride giving acetylenic diketones is best performed in ethereal vent in the presence of CuI (5% mol) and Et3N (2 mmol) in ligand-free conditions. Stephens-Castro reaction of steroidal oxalyl chloride 2 and phenylacetylene 3a in TH the mentioned conditions led to compound 4 in a yield of 9%. Varying amounts of cop iodide (5, 10, 20 mol %) and Et3N (2 or 3 equiv.) and also using the additive (TMEDA equiv.) gave no acceptable results; the alkyne-1,2-dione 4 was obtained in trace amou Further, it was not possible to obtain the target compound 4 when replacing the b with N,N-diisopropylethylamine (DIPEA) or K2CO3, and the solvent with 1,4-diox The best result for the coupling step was obtained by replacing ether solvents with b zene, reducing the amount of Et3N to 1 equiv. and increasing the amount of catalyst t mol %. By performing the reaction at 40 °C, compound 4 was isolated in 48% yield a column chromatography. Increasing the reaction temperature diminishes the yield, prolonging the reaction time (more than 24 h) does not increase the yield. Under found conditions, spirostene ynediones 5-8, containing fragments of aryl acetylenes w 4-methoxy-, 4-fluoro-, 4-ethyl-and 3-methyl substituent, were obtained in 18-48% lated yields. Characteristically, the content of alkyne-1,2-diones 4-8 in the reaction m ture was 70-80% according to 1 H NMR data; however, a significant part of the prod was decomposed during isolation by chromatography on silica gel due to the low sta ity of alkyne-1,2-diones. The structure of (22R,25R)-spirost-5-en-3β-yl 2-o 4-(phenyl)but-3-ynoate 4 has been corroborated by an X-ray structure analysis (Figur   the heterocyclization reaction of the resulting alkynyl diketones [39,40]. Initially, we worked out a three-component synthesis of alkyne-1,2-diones genin 1. Diosgenin 1 was first converted to the steroidal monooxalyl chloride 2 b ing with an excess of oxalyl chloride in chloroform at 0 °C. After removing the e oxalyl chloride and chloroform in vacuo, the resulting 3β-O-(2-chloro-2-ox te)-5-spirostene 2 (Scheme 2) was involved in the Stephens-Castro cross-couplin tion with aryl alkynes (3a-e). It was known that cross-coupling of terminal ace with monooxalyl chloride giving acetylenic diketones is best performed in ether vent in the presence of CuI (5% mol) and Et3N (2 mmol) in ligand-free conditio Stephens-Castro reaction of steroidal oxalyl chloride 2 and phenylacetylene 3a in the mentioned conditions led to compound 4 in a yield of 9%. Varying amounts of iodide (5, 10, 20 mol %) and Et3N (2 or 3 equiv.) and also using the additive (TME equiv.) gave no acceptable results; the alkyne-1,2-dione 4 was obtained in trace am Further, it was not possible to obtain the target compound 4 when replacing t with N,N-diisopropylethylamine (DIPEA) or K2CO3, and the solvent with 1,4-d The best result for the coupling step was obtained by replacing ether solvents w zene, reducing the amount of Et3N to 1 equiv. and increasing the amount of cataly mol %. By performing the reaction at 40 °C, compound 4 was isolated in 48% yie column chromatography. Increasing the reaction temperature diminishes the yie prolonging the reaction time (more than 24 h) does not increase the yield. Un found conditions, spirostene ynediones 5-8, containing fragments of aryl acetylen 4-methoxy-, 4-fluoro-, 4-ethyl-and 3-methyl substituent, were obtained in 18-4 lated yields. Characteristically, the content of alkyne-1,2-diones 4-8 in the reacti ture was 70-80% according to 1 H NMR data; however, a significant part of the p was decomposed during isolation by chromatography on silica gel due to the low ity of alkyne-1,2-diones. The structure of (22R,25R)-spirost-5-en-3β-yl 4-(phenyl)but-3-ynoate 4 has been corroborated by an X-ray structure analysis (Fi   a one-pot alkynylation-heterocyclization sequences that start from diosgenin 1 and apply treatment with oxalyl chloride for formation of 3β-O-(2-chloro-2-oxoacetate)-doiosgenin, Stephens-Castro conditions for the generation of ynediones 4-8 and their reaction with phenylhydrazine (Scheme 3). The stage of heterocyclization was carried out by adding phenyl hydrazine hydrochloride 9 (1 equiv.) to the reaction mixture containing steroidal alkyne-1,2-diones 4-8 and triethylamine (1 equiv.) in benzene. The heterocyclization step, similar to the Stephens-Castro cross-coupling reaction, proceeded in benzene but required an increase in temperature to 60 • C. After 24 h (indicated by TLC), the corresponding 5-aryl substituted 3-O-(pyrazol-3-yloxo)diosgenin derivatives 10-14 were obtained with an isolated yield of 46-60% (Scheme 3). The structures of 3-O-(pyrazolocarbonyl)spirostenes 11,12 were confirmed by X-ray structural analysis ( Figure 2). Importantly, excellent regioselectivity was observed, and only one regioisomer was formed during this one-vessel process.
Based on the analysis of the spectral and analytical data of obtained pyrazol we can suggests that the heterocyclization reaction of alkyne-1,2-diones 4-8 star the nucleophilic addition of phenylhydrazine at the carbonyl group conjugated triple bond to form the corresponding α,β-alkynyl phenylhydrazones, which dergo intramolecular cyclization. Heterocyclization of (hetero)aryl alkyne-1, Inspired by the alkynylation of in situ generated 3β-O-(2-chloro-2-oxoacetate)-5-spirostene 2 and taking into account the low stability of compounds 4-8, we set out to design a one-pot alkynylation-heterocyclization sequences that start from diosgenin 1 and apply treatment with oxalyl chloride for formation of 3β-O-(2-chloro-2-oxoacetate)-doiosgenin, Stephens-Castro conditions for the generation of ynediones 4-8 and their reaction with phenylhydrazine (Scheme 3). The stage of heterocyclization was carried out by adding phenyl hydrazine hydrochloride 9 (1 equiv.) to the reaction mixture containing steroidal alkyne-1,2-diones 4-8 and triethylamine (1 equiv.) in benzene. The heterocyclization step, similar to the Stephens-Castro cross-coupling reaction, proceeded in benzene but required an increase in temperature to 60 °C. After 24 h (indicated by TLC), the corresponding 5-aryl substituted 3-O-(pyrazol-3-yloxo)diosgenin derivatives 10-14 were obtained with an isolated yield of 46-60% (Scheme 3). The structures of 3-O-(pyrazolocarbonyl)spirostenes 11,12 were confirmed by X-ray structural analysis ( Figure 2). Importantly, excellent regioselectivity was observed, and only one regioisomer was formed during this one-vessel process.  Annulation reactions of ynediones have been broadly explored, and these reactions usually need additional catalyst [43,45,46] or high temperature [39]. In our case, the copper(I)/triethylamine-catalyzed synthesis of pyrazoles in yields up to 46-60% was accomplished in one vessel from the reaction of diosgenin 1 with oxalyl chloride at room temperature, with the removal of the solvent from the reaction mixture, diluting with benzene and sequential addition of triethylamine, CuI, aryl alkynes 3a-e, and phenylhydrazine hydrochloride 9.
Based on the analysis of the spectral and analytical data of obtained pyrazoles 10-14 we can suggests that the heterocyclization reaction of alkyne-1,2-diones 4-8 started with the nucleophilic addition of phenylhydrazine at the carbonyl group conjugated with the triple bond to form the corresponding α,β-alkynyl phenylhydrazones, which then undergo intramolecular cyclization. Heterocyclization of (hetero)aryl alkyne-1,2-diones with N-acylhydrazines with the formation of pyrazole derivatives was performed in a medium of protic solvents-methanol or 2-methoxyethanol [38,40]. We also attempted to Annulation reactions of ynediones have been broadly explored, and these reactions usually need additional catalyst [43,45,46] or high temperature [39]. In our case, the copper(I)/triethylamine-catalyzed synthesis of pyrazoles in yields up to 46-60% was accomplished in one vessel from the reaction of diosgenin 1 with oxalyl chloride at room temperature, with the removal of the solvent from the reaction mixture, diluting with benzene and sequential addition of triethylamine, CuI, aryl alkynes 3a-e, and phenylhydrazine hydrochloride 9.
It is noteworthy that in mild reaction conditions by using of 2-methoxyethanol as the solvent we were able to isolate alkynyl substituted steroidal (E)-hydrazones 15-19, in several examples, as the main products. We found that (Z)-hydrazone 20 has been not so stable in the reaction conditions; on heating to 40-60 °C in benzene, or on silica gel during chromatography, this compound partially converted into the stable (E)-izomer 15. The structures of steroidal (E)-and (Z)-phenylhydrazones 15,20 and minor 2,3,5-trisubstituted pyrazole 21 were confirmed by the data of X-ray structural analysis ( Figure 3). PhH-EtOH, 1:2, 60 °C (b) Results of employing various solvents in the stage of ynedione-phenylhydrazine interaction. Table 1. The effect of the solvent on the yield of products in the stage of ynedione-phenylhydrazine interaction.
It must been noted that α,β-alkynic hydrazones are an important class with practical value, they have been emanated as powerful synthons for constructing diverge ranges of cyclic compounds through transition metal catalyzed or transition metal free reactions [47][48][49].  It must been noted that α,β-alkynic hydrazones are an important class with practical value, they have been emanated as powerful synthons for constructing diverge ranges of cyclic compounds through transition metal catalyzed or transition metal free reactions [47][48][49].
The sequential four-component reaction of diosgenin 1, oxalyl chloride, phenylacetylene 3a, and hydrazine monohydrate 22 occurred smoothly and led to the formation of 3,5-disubstituted pyrazole 23 (Scheme 5, Figure 4). The final step of this sequence consisted of Michael addition/cyclocondenzation/elimination reactions, and ran in 2-methoxyethanol at room temperature.   The sequential four-component reaction of diosgenin 1, oxalyl chloride, phenylacetylene 3a, and hydrazine monohydrate 22 occurred smoothly and led to the formation of 3,5-disubstituted pyrazole 23 (Scheme 5, Figure 4). The final step of this sequence consisted of Michael addition/cyclocondenzation/elimination reactions, and ran in 2-methoxyethanol at room temperature. It must been noted that α,β-alkynic hydrazones are an important class with practical value, they have been emanated as powerful synthons for constructing diverge ranges of cyclic compounds through transition metal catalyzed or transition metal free reactions [47][48][49].
The sequential four-component reaction of diosgenin 1, oxalyl chloride, phenylacetylene 3a, and hydrazine monohydrate 22 occurred smoothly and led to the formation of 3,5-disubstituted pyrazole 23 (Scheme 5, Figure 4). The final step of this sequence consisted of Michael addition/cyclocondenzation/elimination reactions, and ran in 2-methoxyethanol at room temperature.   It must been noted that α,β-alkynic hydrazones are an important class with practical value, they have been emanated as powerful synthons for constructing diverge ranges of cyclic compounds through transition metal catalyzed or transition metal free reactions [47][48][49].
The sequential four-component reaction of diosgenin 1, oxalyl chloride, phenylacetylene 3a, and hydrazine monohydrate 22 occurred smoothly and led to the formation of 3,5-disubstituted pyrazole 23 (Scheme 5, Figure 4). The final step of this sequence consisted of Michael addition/cyclocondenzation/elimination reactions, and ran in 2-methoxyethanol at room temperature.   N-Acyl substituted hydrazones, as expected, showed a softer character of the nucleophilic center, which is expressed in their initial addition at the soft electrophilic center of the triple bond of alkynyl ketones with the formation of intermediate alken-hydrazide A and their subsequent intramolecular cyclization leading to 1-hydroxy-4,5-dihydro-1Hpyrazoles [37]. The reactions of the in situ formed alkynyldiketones with N-acylhydrazides usually need an additional catalyst or high temperatures [40]. We found, that the consecutive four-component reaction of diosgenin 1, oxalyl chloride, phenylacetylene 3a, and hydrazides of benzoic or 4-bromobenzoic acids 24a,b led to the formation of 3-O-(1-aroyl-5hydroxy-3-phenyl-4,5-dihydro-1H-pyrazoline)-spirostenes 25 or 26 isolated in the yield of 49 and 42%, respectively (Scheme 6). Thus, the initial reaction of N-acylhydrazines with the in situ formed steroidal alkynyldione proceeds through the addition of the nucleophile at the triple bond and the formation of intermediate alken-hydrazide A. Additionally, in the reaction of 1 with 4-bromobenzoic acid hydrazide 24b, the aromatizing elimination of water and simultaneous deacylation of 4-bromophenacyl substituent furnishing of compound 23 ( Figure 4) was observed (yield 18%).
The structures of synthesized compounds were established by 1 H NMR and 13 C, and IR spectroscopies, mass spectrometry, and elemental analysis data. The 1 H NMR spectrum of alkynyl-substituted (E)-hydrazones 15-19 in deuterochloroform is characterized by the presence of an NH-proton signal in the form of a broad singlet at 9.05-9.09 ppm, while the signal of a similar proton of (Z)-hydrazone 20 appears as a singlet at δ12.80 ppm. Two alkynic carbons (C-3′ and C-4′), in the spectra of alkynyl substituted (Z)-hydrazone 20 resonate closely at δ 85.3 and 90.0 ppm (Δδ 4.7 ppm). In the (E)-isomers 15-19, the alkynic carbon adjacent to the carbonyl group is comparatively upfield (δ 77.3-77.8 ppm), while the other alkynic carbon (C-4′) is relatively downfield (δ 103.6-105.2 ppm) and the chemical shift difference between these carbons is roughly 26.1−28.3 ppm. In brief, the absolute value of chemical shift difference between alkynic carbons in the (E)-isomer is typically bigger as compared to that in the (Z)-isomer. A characteristic down-field shift was observed for the C-2′ carbon atom (δ 117.6-118.2 ppm) for α,β-alkynyl-substituted (E)-hydrazones 15-19 as compared to the signal for (Z)-isomer (δ 113.9 ppm). The diastereotopic protons H-4′ in the 1 H NMR spectra of pyrazolines 25 and 26 manifested as doublets at δ 3.40, 3.60 (25) and 3.43, 3.60 ppm (26) (J = 17.9 Hz), respectively. The formation of a mixture of diastereomeric 5-hydroxypyrazolines is indicated by the doubling of some signals in the 13 C NMR spectrum; the largest difference was observed for the chemical shifts of carbon atom C-5′ (δ 89.5; 89.6 ppm) (Supplementary Materials, 1 H and 13 C NMR spectra of compounds 25 and 26; P. [39][40][41][42]. The structure of compounds 4, 11, 12, 15, 20, 21 and 23 were determined by single crystal X-ray analysis (Figures 1-4). The analysis of the molecular geometries was performed using the PLATON program [50,51]. All the compounds contain the spirosten six-ring moiety: the tetrahydropyran cycle is spiro fused with the furan cycle, which is cis-fused with the cyclopentan ring, and the rest are trans-fused in all the compounds Scheme 6. One-pot synthesis of steroidal 1-aroyl-5-hydroxypyrazolines 25,26.
The structures of synthesized compounds were established by 1 H NMR and 13  The structure of compounds 4, 11, 12, 15, 20, 21 and 23 were determined by single crystal X-ray analysis (Figures 1-4). The analysis of the molecular geometries was performed using the PLATON program [50,51]. All the compounds contain the spirosten six-ring moiety: the tetrahydropyran cycle is spiro fused with the furan cycle, which is cis-fused with the cyclopentan ring, and the rest are trans-fused in all the compounds observed. The cyclohexane and pyran cycles adopt a chair conformation, the cyclohexene-a halfchair conformation. The furan cycle has an envelope conformation with the deviation of the O1 atom from the rest of the atoms of the cycle in 4, 12, and C22 in 15. The different deviating atom in the furan cycle of 15 causes the absence of the intramolecular hydrogen bond C26-H26A . . . O1 occurred in 4, 11 and 12, with parameters shown in Table 2. The cyclopentane ring has a twist conformation in 4, 11, 12, and an envelope in 15. The intramolecular hydrogen bond C3-H3 . . . O4 keeps atom O4 almost in the plane of C3O3C1 in all compounds.   (Table 1), that leads to an almost plane substituent in C3. Inter-plane angles of the hydrogen bond cycle O4=C1 C2 =N1 N2 -H2 with phenyl rings C1 ÷ C6 and C1 ÷ C6 are 6.85 and 10.74 accordingly; that is less that in 15. The bond lengths distribution of O4C1 C2 =N1 N2 C1 moiety is also different for 15 and 20 (Table 3). The common feature of 11, 12, 21 and 23 structures is that C1 (=O4)O3 moiety lays practicaly in the pyrazol plane that can be convenient to form common conjugated π-system. Thus, the torsion angle N1 C5 C1 O3 is −9.9(4) o (11), −11.2(3) o (12), −9.8(4) and −1.0(5) o for two independent molecules of (21) and 0.7(5) o (23).

Anti-Inflammatory Activity of Diosgenin 1 and Its 3-O-Substituted Derivatives in the Histamine-Induced Paw Edema Model
As previously mentioned, diosgenin 1 is a multi-targeted agent that has immense potential to be used as a wonder drug for the treatment of innumerable chronic diseases, such as cancer, CVDs, metabolic and nervous system disorders, and different types of inflammatory diseases [52,53]. Diosgenin 1 and its 3-O-substituted derivatives 7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamineinduced paw edema model [54]. The results are presented in Table 4. Table 4. Anti-inflammatory activity of diosgenin 1 and its 3-O-substituted derivatives in the histamineinduced paw edema model.

Histamine-Induced Paw Edema Model
As previously mentioned, diosgenin 1 is a multi-targeted agent that has immense potential to be used as a wonder drug for the treatment of innumerable chronic diseases, such as cancer, CVDs, metabolic and nervous system disorders, and different types of inflammatory diseases [52,53] . Diosgenin 1 and its 3-O-substituted derivatives  7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamine-induced paw edema model [54]. The results are presented in Table 4. As previously mentioned, diosgenin 1 is a multi-targeted agent that has immense potential to be used as a wonder drug for the treatment of innumerable chronic diseases, such as cancer, CVDs, metabolic and nervous system disorders, and different types of inflammatory diseases [52,53] . Diosgenin 1 and its 3-O-substituted derivatives  7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamine-induced paw edema model [54]. The results are presented in Table 4. potential to be used as a wonder drug for the treatment of innumerable chronic diseases, such as cancer, CVDs, metabolic and nervous system disorders, and different types of inflammatory diseases [52,53]. Diosgenin 1 and its 3-O-substituted derivatives 7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamine-induced paw edema model [54]. The results are presented in Table 4. 10 R = potential to be used as a wonder drug for the treatment of innumerable chronic diseases, such as cancer, CVDs, metabolic and nervous system disorders, and different types of inflammatory diseases [52,53]. Diosgenin 1 and its 3-O-substituted derivatives 7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamine-induced paw edema model [54]. The results are presented in Table 4. 13 R = such as cancer, CVDs, metabolic and nervous system disorders, and different types of inflammatory diseases [52,53]. Diosgenin 1 and its 3-O-substituted derivatives 7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamine-induced paw edema model [54]. The results are presented in Table 4. 14 R = inflammatory diseases [52,53]. Diosgenin 1 and its 3-O-substituted derivatives 7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamine-induced paw edema model [54]. The results are presented in Table 4.  7,8,10,13-16,18,19,21,23 and 26 were tested for their anti-inflammatory activities using the histamine-induced paw edema model [54]. The results are presented in Table 4. histamine-induced paw edema model [54]. The results are presented in Table 4.   Table 4 illustrate that diosgenin 1, alkynyldiketones 7,8, hydrazones 15-19 Table 4 illustrate that diosgenin 1, alkynyldiketones 7,8, hydrazones 15-19, and pyrazole conjugate 23, administered orally in mice (50 mg/kg dose), decreased the paw edema by 29-59 percent in relation to the control group. The same attribute of the 23 Table 4 illustrate that diosgenin 1, alkynyldiketones 7,8, hydrazones 15-19, and pyrazole conjugate 23, administered orally in mice (50 mg/kg dose), decreased the paw edema by 29-59 percent in relation to the control group. The same attribute of the reference drug diclofenac sodium in the effective dose of 10 mg/kg was 56 percent. Diosgenin-pyrazole conjugate 23 showed the highest activity. However, substituent on the 24 Table 4 illustrate that diosgenin 1, alkynyldiketones 7,8, hydrazones 15-19, and pyrazole conjugate 23, administered orally in mice (50 mg/kg dose), decreased the paw edema by 29-59 percent in relation to the control group. The same attribute of the reference drug diclofenac sodium in the effective dose of 10 mg/kg was 56 percent. Diosgeninpyrazole conjugate 23 showed the highest activity. However, substituent on the nitrogen atoms in the pyrazole ring led to a decrease in anti-inflammatory activity (10, 13, 14, 21,  26). The activity of alkynyldiketones 7,8, hydrazones 15,18 and 3,5-disubstituted pyrazole derivative 23 compares favorably with that of the starting compound 1. These data indicate that the anti-inflammatory activity of diosgenin derivatives is sensitive to the nature of substitution at the 3-hydroxy substituent and confirms the potential value of this class of biological active substances.

Molecular Docking
The literature search resulted in many in vitro, in vivo and clinical trials that reported the efficacy of diosgenin and its analogs in modulating important molecular targets and signaling pathways, such as PI3K/AKT/mTOR, JAK/STAT, NF-κB, and NFkBp65, which play a crucial role in the development of most of the chronic diseases [53]. It can modulate antioxidant defense and decrease oxidative stress damage [55] and exhibit inhibitory effects on superoxide anion production through the blockade of cAMP, PKA, cPLA2, PAK, Akt and MAPKs signaling pathways [56]. There are three main cellular components involved in the regulation of antioxidant response, and they are Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and antioxidant response elements (ARE). It is now intensively studied that activation of Keap1-Nrf2-ARE signaling can provide protection against various stress and inflammation related diseases, including neurogenerative diseases, autoimmune diseases, and cardiovascular disorders [57]. From the molecular docking assay results, the parent saponin dioscin (glycoside form of diosgenin) showed powerful affinities towards to Sirt1, Keap1 and NF-κBp65, indicating that the compound may directly bind to these proteins, to exert its biological activities [58]. In this study we aimed to provide in silico evidence that pyrazole-diosgenin hybrids bind Keap1 and hence could be employed as promising Nrf2 activators. AutoDock Vina 1.5.6 was used to perform molecular docking of diosgenin 1, and its derivatives 15 and 23.
The tunnel-like binding site of the Kelch domain is substantially hydrophilic. It has a fairly limited size. We can also observe that large hydrophobic diosgenin scaffold 1 (−3.169 kcal/mol) cannot penetrate deep into the binding site ( Figure 5A). However, the introduction of a substituent at the C-3 position of the diosgenin structure significantly reduces the estimated minimal binding energy of the steroidal α,β-alkynyl (E)-hydrazone 15 (−6.344 kcal/mol) and pyrazole 23 (−4.828 kcal/mol) to the Kelch domain, which is probably associated with the ability of the substituents to interact with amino acids deep in the binding site ( Figure 5B,C). The superposition of structures in the binding site indicates that the diosgenin scaffolds of the studied molecules remain in the wide outer part of the binding site. Functionalization of the hydroxyl group of the diosgenin ring A leads to a very close orientation of the diosgenin scaffold of derivatives in the space of the binding site ( Figure 5D). The structures of the substituents of the new diosgenin derivatives are stabilized in the deep part of the binding site due to hydrophobic interactions. Attention is drawn to the formation of a hydrogen bond of the same type for compounds 15 and 23 with an amino acid residue Ser602. The hydroxyl group of diosgenin 1 can probably form a hydrogen bond with the amino acid residue Ser508.
part of the binding site. Functionalization of the hydroxyl group of the diosgenin ring A leads to a very close orientation of the diosgenin scaffold of derivatives in the space of the binding site ( Figure 5D). The structures of the substituents of the new diosgenin derivatives are stabilized in the deep part of the binding site due to hydrophobic interactions. Attention is drawn to the formation of a hydrogen bond of the same type for compounds 15 and 23 with an amino acid residue Ser602. The hydroxyl group of diosgenin 1 can probably form a hydrogen bond with the amino acid residue Ser508.    The 1 H spectra for compounds 7, 11, 12 were obtained on 'Bruker AV 400 , for 8, 2on 'Bruker AV 300 , for 21-on 'Bruker AV 600 , 13 C spectra for 7, 8, 11, 12, 20, 21-on 'Bruker DRX-500 . Deuterochloroform (CDCl 3 ) was used as a solvent, with residual CHCl 3 (δ H = 7.24 ppm) or CDCl 3 (δ C = 77.0 ppm) being employed as internal standards. NMR signal assignments were carried out with the aid of a combination of 1D and 2D NMR techniques that included 1 H, 13 C, COSY, HSQC, and HMBC spectra. IR absorption spectra were recorded on a Vector 22 FT-IR spectrometer in KBr pellets. The specific rotation values [α] D were obtained on a PolAAr 3005 polarimeter. Melting points were determined using termosystem Mettler Toledo FP900 (Columbus, OH, USA). HRMS spectra were recorded on a DFS mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), evaporator temperature 180-220 • C, EI ionization at 70 eV). Elemental analysis was carried out on a 1106 Elemental analysis instrument (Carlo-Erba, Milan, Italy). The X-ray diffraction experiments for crystals of 4, 11, 12, 15, 20, 21 and 23 were performed at ambient conditions on a Bruker KAPPA APEX II diffractometer (graphite-monochromated Mo Kα radiation). Reflection intensities were corrected for absorption by SADABS program [59]. The structure was solved by direct methods using the SHELXS-97 program and refined by an anisotropic (isotropic for all H atoms) full-matrix least-squares method against F 2 of all reflections by SHELX-97 [60]. The positions of the hydrogen were calculated geometrically and refined in a riding model.

Chemistry
The reaction progress and the purity of the obtained compounds were monitored by TLC on Silufol UV-254 plates (CHCl 3 -EtOH, 9:1; detection under UV light or by treatment with iodine vapor). The reaction progress and the purity of the obtained compounds were monitored by TLC on Silufol UV-254 plates (Kavalier, Czech Republic, CHCl 3 -EtOH, 100:1; detection under UV light or by spraying the plates with a 10% water solution of H 2 SO 4 followed by heating at 100 • C). Products were isolated by column chromatography on silica gel 60 (0.063-0.200 mm, Merck KGaA, Darmstadt, Germany), eluting with indicated solvent systems. The chemicals used: arylacetylenes 3a-e, oxalyl chloride, phenyl hydrazine hydrochloride 9, hydrazine monohydrate 22, benzhydrazide 24a and 4-bromobenzhydrazide 24b, were purchased from Aldrich (St. Louis, MO, USA) or Alfa Aesar (GmbH, Karlsruhe, Germany). Solvents (CHCl 3 , 2-methoxyethanol, ethanol, benzene) and Et 3 N were purified by standard methods and distilled under a stream of argon just before use. Copies of NMR spectra ( 1 H and 13  One-Pot Synthesis of Steroidal Ynediones (4)(5)(6)(7)(8) A solution of diosgenin (800 mg, 1.93 mmol) in CHCl 3 (10 mL) was added dropwise to a cold stirred solution of oxalyl chloride (980 mg (0.66 mL), 7.72 mmol) in CHCl 3 (5 mL) in argon atmosphere at 0 • C for 1 h and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure, the residue was treated with CHCl 3 (3mL) and additionally evaporated. This procedure was repeated three times for removing the trace of oxalyl chloride and diluted with C 6 H 6 (10 mL) in an argon flow. The corresponding aryl acetylene 3a-e (1.00 mmol), CuI (19 mg, 0.1 mmol) and Et 3 N (0.138 mL, 1.00 mmol) were added subsequently in an argon flow at room temperature. The mixture was heated under stirring at 40 • C for 12 h (TLC), after that solvent was removed under reduced pressure, and the residue was purified by column chromatography (eluent petroleum ether-ether, 20:1) to give compounds (4)(5)(6)(7)(8).

Conclusions
We report the sequential one-pot synthesis, including the steps of acylation of the alcohol substrate diosgenin with oxalyl chloride, the Stephens-Castro reaction of the formed 3-O-(2-chloro-2-oxoacetyl)diosgenin with arylalkynes and selective heterocyclization of alkynyldiones with hydrazines and hydrazides. We found the conditions of selective one-pot formation of 5-aryl substituted 3-O-(pyrazol-3-yloxo)diosgenin derivatives. By using 2-methoxyethanol at the step of reaction of spirostene alkyne-1,2-diones with phenylhydrazine hydrochloride regioisomeric α,β-alkynic hydrazones were also isolated. Carrying out the heterocyclization step of ynedione with hydrazine monohydrate in 2methoxyethanol allowed the synthesis of 5-phenyl substituted steroidal pyrazole, which was found to exhibit high anti-inflammatory activity, comparable to that of diclofenac sodium. By in silico experiments, it was shown that the obtained steroidal α,β-alkynyl (E)-hydrazones and a diosgenin-pyrazole conjugate are incorporated into the binding site of the protein Keap1 Kelch-domain by their substituent at the hydroxyl group and form more non-covalent bonds, and have higher affinity than the initial spirostene core. It is foreseeable that the described one-pot consecutive process will find broad application in drug discovery and other related research fields.